Tumor necrosis factor-.alpha. (TNF-.alpha.), also known as cachectin, is a multifunctional cytokine produced mainly by activated macrophages. In vitro, it has diverse biological effects (reviewed by Manogue et Cerami, 1988) including killing of transformed cells (Carswell et al., 1975), stimulation of granulocytes and fibroblasts (Old, 1985; Vilchek et al., 1986; Beutler and Cerami, 1987), damage to endothelial cells (Sato et al, 1986), and anti-parasitic effects (Taverne et al., 1984). In vivo, it plays a key role as an endogenous mediator of inflammatory, immune and host defence functions and it is involved in a number of pathological conditions in man and mouse such as septic shock, cachexia, capillary leak syndrome, hemorhagic necrosis of multiple organs, etc. It is capable of acting independently and in conjunction with other factors affecting a whole plethora of different body functions. These effects can either be beneficial or life-threatening to the host. Some of these effects are direct, others may be mediated via the induction of other secreted factors. The biological effects of TNF-.alpha. are mediated via binding to specific cell surface receptors.
The primary structure of human and mouse TNF-.alpha. (Pennica et al., 1984; Fransen et al., 1985) and of two different TNF-receptors (p55-TNF-R and p75-TNF-R) has been deduced from the nucleotide sequence of the cloned cDNA (Dembic et al., 1990; Loetscher et al., 1990). Both receptors bind not only TNF-.alpha. but also TNF-.beta. or lymphotoxin with high affinity (Schoenfeld et al., 1991). TNF-.beta. is a related lymphocyte product (TNF-.alpha. and TNF-.beta. share 32% homology on the amino acid sequence level) that exhibits pleiotropic activities very similar to those of TNF-.alpha.. X-ray crystallographic analysis revealed that the tertiary structure of both molecules is virtually identical except that the TNF-.beta. trimer creates a molecule that is less elongated than the TNF-.alpha. trimer and the latter has a top-region that flares open (Eck et al, 1992).
Besides the cell receptor interaction, TNF-.alpha. has been shown to have a lectin-like property for the oligosaccharide ligands chitobiose and Man(.alpha.1,3)-(Man(.alpha.1,6)-Man (Hession et al., 1987; Sherblom et al., 1988). They further demonstrated that the TNF-.alpha. protein has at least two different binding sites, one lectin-like and the other directed at cell surface receptors. Several TNF-.alpha. mutants were described for which the binding to the cellular TNF-receptor p55 and/or p75 was hampered. All these mutations are located in the lower half of the pyramidal structure of the biologically active TNF-.alpha. trimer (Van Ostade et al., 1991; Van Ostade et al., 1992; EP-A-0 486 908).
The availability of recombinant TNF has enhanced its use in tumor therapy. However, the in vivo tumoricidal effects of TNF-.alpha. have always been accompanied by toxic side effects. Different approaches were followed to overcome these noxious effects, including the use of monoclonal antibodies as well as fragments thereof, which neutralize the in vitro and in vivo toxic properties of TNF-.alpha. (e.g. EP-A-0 350 690).
In addition to its immunomodulating activity, TNF-.alpha. has also been shown to be involved in the control of growth and differentiation of various parasites. Upon infection of the host, parasites are capable of inducing the secretion of different cytokines such as TNF which may affect the course of the disease. For instance, in the case of malaria, TNF-.alpha. can be protective in certain circumstances, such as inhibiting parasite survival in rodent malaria (Clark et al., 1987; Taverne et al., 1987). By contrast, its overproduction can be detrimental to the host and can contribute to the pathology of the disease (Clark, 1987; Grau et al., 1989).
In schistosomiasis, the parasite uses the host-derived immunoregulatory protein TNF-.alpha. as a signal for replication and transmission (Amiri et al., 1992). Moreover, the authors found that the parasite worms required TNF-.alpha. for egg laying and for excretion of eggs from the host. In cutaneous leishmaniasis, TNF-.alpha. a plays a protective role (Titus et al., 1989; Liew et al., 1990).
African trypanosome species are the etiological agents for African trypanosomiasis, or sleeping sickness, and are a major cause of both human disease (Trypanosoma brucei rhodesiense and T. brucei gambiense) and cattle disease (T. brucei brucei, T. evansi, T. vivax, T. congolense) in Africa. Injected into their mammalian hosts by tsetse flies, trypanosomes remain extracellular bloodstream trypomastigotes throughout their mammalian phase. In trypanosomiasis, it has been shown that rabbits infected with Trypanosoma brucei brucei developed cachexia and suppression of lipoprotein lipase levels (Rouzer and Cerami, 1980). These parameters are indicators of TNF-.alpha. bioactivity (Beutler et al., 1985), thus suggesting that the parasite infection induced TNF in vivo. Kongshavn et al. (1988) reported that TNF-.alpha. inhibited the growth of in vitro cultures of Trypanosoma musculi, but administration of TNF-.alpha. in vivo during the course of infection led to a net increase in parasite development. The in vitro data indicate that a direct interaction between TNF-.alpha. and T. musculi can take place. All these experiments point to a direct interaction between host-derived TNF-.alpha. and parasites influencing the course and the outcome of parasitemia.
Considerable evidence implicates TNF-.alpha. is the principle mediator of endotoxic or septic shock (Cerami and Beutler, 1988). The intravenous injection of LPS (or endotoxin) in animals produces several parameters of the septic shock syndrome, namely, hypotension, decreased systemic vascular resistance, leukopenia, thrombocytopenia, and tissue dammage. In experimental animals, TNF-.alpha. produces hypotension, leukopenia, and local tissue necrosis (Okusawa et al., 1988). Administration of anti-TNF-.alpha. antibodies to baboons (Tracey et al., 1987) or rabbits prevents the shock induced by endotoxin (Mathison et al., 1988).
The term "TNF-induced septic shock" refers to the shock that develops in the presence of severe infection, especially following bacteremia with Gram-negative bacteria and release of endotoxin. It may also be caused by any class of microorganism, including Gram-positive bacteria, viruses, fungi, protozoa, spirochetes and rickettsiae. The pathophysiology of septic shock is very complex (see Harrison's Principles of Internal Medicine, 12th ed, McGraw-Hill, New York, pp. 502-507). Death is caused by progression to multiple organ failure and circulatory collapse.
Sherblom et al., (1988) have shown that the TNF-.alpha. has a lectin-like affinity for the glycoprotein uromodulin. Interestingly, this glycoprotein is able to potently inhibit the TNF-induced shock in vivo, whereas it does not block the tumoricidal activity of TNF-.alpha. on L929 cells in vitro. The authors suggested therefore that the lectin-like region of TNF-.alpha. is implicated in the toxic effects of TNF-.alpha. in septic shock, but not in its tumoricidal activity.